【作者】 邵铁梅；

【导师】 黄大昉； 刘大群；

【作者基本信息】 河北农业大学 ， 植物病理学， 2005， 博士

【摘要】 苏云金芽孢杆菌(Bacillus thuringiensis简称Bt)是一类分布极其广泛的革兰氏阳性细菌,在特定生理阶段产生芽孢,并形成具有杀虫活性的伴孢晶体。随着对Bt研究的不断深入,又发现了许多与杀虫相关的其它有效成分,如辅助蛋白(Helper protein)、营养期杀虫蛋白(Vegetative Insecticidal Proteins,VIPs)和增效物质Zwittermicin A。Zwittermicin A是一种新型广谱抗生素,该抗生素可抑制多种真核微生物和原核微生物,其中包括多种植物病原菌,而且与Bt毒素共同使用能显著提高其杀虫活性。本文对国内一批Bt菌株进行了筛选;对影响Zwittermicin A生成和活性的条件进行了探索;对Bt菌株G03的Zwittermicin A合成基因簇进行了克隆、分析、及部分开放阅读框的功能验证。 采用PCR与抑菌活性相结合的方法对国内196株Bt菌株进行筛选,有67株菌有抑菌活性,其中21株抑菌活性明显高于其余的46株。 Bt菌株G03高产Zwittermicin A,有很高的抑菌活性。选用营养程度不同的培养基对Bt菌株G03进行培养、结合菌体观察和培养液上清液活性测定,证明Zwittermicin A的生成与芽孢的产生无依赖关系:通过测定超声波处理前后的上清液的抑菌活性差异,证明Zwittermicin A的生成和分泌之间有时间的延迟;并测定了不同pH值对Zwittermicin A抑菌活性的影响,发现其在中性偏碱的环境中活性较高。 从G03克隆了zmaR全长基因,并完成了序列测定。该基因编码区为1125bp,编码的蛋白质由375个氨基酸残基组成,分子量为43.5kDa,等电点pI4.95。通过载体pET-21b将zmaR基因导入大肠杆菌BL21,该基因可正常表达,使宿主菌BL21获得对Zwittermicin A的抗性。 构建了Bt菌株G03基因组的BAC文库,该文库包含4713个克隆,平均插入片断大小为73kb,该文库相当于59个库容量。采用PCR方法对该库的1000个克隆进行筛选,得到4个阳性克隆,其中两个阳性克隆(插入片段分别为65kb和94kb)包含已发表的16kb Zwittermicin A合成基因簇(发表的16kb序列为部分Zwittermicin A合成基因簇)。 对插入片段为65kb的阳性克隆进行测序、分析,得到36.665kb的序列,共包括15个开放阅读框,在已发表的Zwittermicin A合成基因簇基础上又发现了完整的orf9,和5个新的开放阅读框,3个位于已发表序列的上游,命名为orfA、DrfB、orfC,2个位于已发表序列的下游,命名为orfN和orfO。新发现的开放阅读框编码的蛋白与聚酮类抗生素和多肽类抗生素合成酶有较高的同源性,Zwittermicin A的合成可能是聚酮类抗生素和多肽类抗生素的杂合途径。 对orfC的功能基因进行了验证,通过重叠PCR方法构建了orfC的突变盒,插入温度敏感的E.coli-Bt穿梭载体pRN5101得到突变质粒pRNC,导入产Zwittermicin A的Bt菌株218,经高温突变获得orfC突变株,突变株丧失了抑菌活性。将orfC全长基因导入突变株后,又使其恢复了抑菌活性,表明orfC是Zwittermicin A合成必需的。 本研究为进一步开展Zwittermicin A同Bt增效作用及其分子机制的研究提更多还原

【Abstract】 Bacillus thuringiensis is an ubiquitous Gram-positive, spore-forming bacterium, which is well-known and widely used as biopesticide. Its insecticidal activity is largely attributed to the formation of parasporal crystals, which are formed during the stationary phase of the B. thuringiensis growth cycle. Many B. thuringiensis isolates also produce other virulent factors, such as helper protein, secreted vegetative insecticidal proteins (VIPs), and Zwittermicin A. Zwittermicin A, a novel, broad-spectrum, aminopolyol antibiotic, is produced by Bacillus cereus and B. thuringiensis strains. It has activity against Oomycetes and their relatives, algae, protists, and certain Gram-negative and Gram-positive bacteria, as well as many plant pathogenic fungi. Zwittermicin A can significantly enhance the insecticidal activity of B. thuringiensis toxins. The main contents of this study included: screening 196 B. thuringiensis by combination PCR analysis with inhibition assay; studing on the condition infected the production of Zwittermicin A and its activity to Erwinia hebicola; Cloning and analysis of the Zwittermicin A biosynthesis cluster.Among 196 B. thuringiensis isolates which were identified by combination PCR analysis with inhibition assay, 67 isoaltes inhibited the growth of Erwinia, and 21 of them were highly active to Erwinia hebicola.B. thuringiensis isolate G03 produces Zwittermicin A, and highly inhibited the growth of Erwinia. Combination cultures in different medium, spores obersevation with inhibition assay, we found that accumulation of Zwittermicin A was not relative to the formation of the spores. Supernatants after supersonic treated have higher activity to Erwinia hebicola than that before, suggesting there was a delay between production and secretion of Zwittermicin A. The results of the pH impact on activity of Zwittermicin A indicated that Zwittermicin A was more active against Erwinia herbicola at pH 7.0-9.0 than at pH 5.5-6.5.zmaR gene was cloned from the G03 isolate and sequenced later. Sequence analysis showed that this gene contained 1125bp, encoded 375 amino acids. Molecular weight of the peptide was 43.5 kDa, and the isoelectric point was pI4.945. This zmaR gene更多还原